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First published online March 6, 2009
doi: 10.1242/10.1242/dev.027920

Development 136, 1127-1135 (2009)
Published by The Company of Biologists 2009
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Target recognition at the tips of postsynaptic filopodia: accumulation and function of Capricious

Hiroshi Kohsaka1 and Akinao Nose1,2,*

1 Department of Complexity Science and Engineering, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8561, Japan.
2 Department of Physics, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.


Figure 1
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  		Fig. 1. CAPS accumulates at the tips of myopodia. (A) CAPS-YFP expressed on M12 strongly accumulates at the tips of myopodia (arrowheads) at 13:00 hours AEL. Inset is a higher magnification view of myopodia. (B-B'') Membrane-bound CFP (B,B''; mCFP; magenta) distributes uniformly along the length of myopodia of M12, in contrast to the clustering of CAPS-YFP (B',B''; green) at the tips. (C) Immunohistochemical visualization of intact CAPS expressed on M12 at 14:30 hours. Whereas most myopodia have vanished due to the fixation process, there is an accumulation of CAPS at the tips of a remaining myopodium (arrowhead). (D) Concurrent visualization of CAPS-GFP by fluorescence and muscle contours by DIC. (E) Cross-section of the muscle and a myopodium cut along the plane indicated by arrowheads in D. The myopodia extend along the interior side of M13. (F,G) Schematic of the trajectories of M12 myopodia in relation to the path of MN12s at 13:00 (F) and 15:00 (G) hours. Blue circles represent CAPS at the tips. In, interior; Ex, exterior side of an embryo. Scale bars: 10 µm in A,C; 4 µm in inset of A,B; 5 µm in D,E.

 

Figure 2
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  		Fig. 2. CAPS-GFP accumulation at the tips is independent of motoneuronal innervation. (A,C) Staining with the axonal marker anti-Fasciclin 2 in wild type (A) and pros mutants (C) at 14:00 hours. Growth cones of motoneurons arrive at ventral muscle field in wild-type embryos (A, arrow). By contrast, motoneurons fail to extend to the target region in pros mutants (C, arrow). (B,D) CAPS-GFP expressed on M12 in wild type (B) and pros mutants (D). CAPS-GFP accumulates at the tips of M12 myopodia in pros mutants as in wild type (B,D; arrowheads). Scale bars: 10 µm in A,C; 5 µm in B,D.

 

Figure 3
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  		Fig. 3. Contacts between muscles and innervating growth cones at the tips of myopodia. (A-A'') The first contact between the muscle and the growth cones occurs at the tips of myopodia. (B-B'') Later interaction between muscles and growth cones also takes place at the tips of myopodia. Time-lapse imaging of axons (as visualized with elav-GAL4: UAS-myristylated-GFP) and myopodia (as visualized with Gal4-5053A: UAS-myristylated-GFP) in an intact embryo, showing three images at 2-minute intervals beginning at 13:00 (A-A'') and 13:16 (B-B'') hours. Time elapsed from the first image is shown in minutes. Arrowheads: myopodia that did (white) or did not (gray) contact the growth cones. g, growth cones; m, M12. Scale bar: 5 µm.

 

Figure 4
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  		Fig. 4. Stabilized and lost contacts between myopodia and growth cones. (A) Fate of contacts between myopodia and growth cones is traced by time-lapse imaging with 2-minute intervals that spans the entire period of synaptogenesis (13:17-14:45 hours). Time elapsed from the first image is shown in minutes. The fate of two contacts was traced (arrowheads). The upper one was established after 8 minutes and was finally stabilized (88 minutes). The lower one, which formed after 2 minutes, was later destabilized (16 minutes). Arrowheads: myopodia that did (white) or did not (gray) contact the presynaptic growth cones. (B) Distribution of the duration of lost contacts between myopodia and growth cones. g, growth cones; m, M12. Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. Myopodial contacts with non-partner motoneurons are eliminated. (A,B) Time-lapse imaging of myopodia and growth cones in intact embryos beginning at 13:45 (A) and 13:55 (B) hours. By this time, the growth cones of MN12s (asterisks) have moved to the proximal edge of M12 and can be largely distinguished from those of MN13s (arrows) that are situated near the proximal edge of M13 (contours of M13 are shown by purple lines). Here, we define neural processes situated on the proximal half of M13 (the boundary of the proximal and distal halves of M13 is indicated by a dotted line in A) as putative MN13 growth cones. The fate of two contacts between M12 myopodia and putative MN13 growth cones is followed. Arrowheads indicate myopodia that did (yellow) or did not (white) contact the growth cones. Both of these contacts are eventually eliminated. Time elapsed from the first image is shown in minutes. (C-C'') Schematic of the positions of growth cones of MN12s and MN13s. (C) At 13:00 hours, growth cones of MN12s and MN13s cannot be distinguished because both are situated near the proximal edge of M13. (C') Slightly later, the distal movement of MN12 growth cones makes it possible to discern them from those of MN13s (arrow). MN13s have begun to arborize along the proximal edge of M13 by this stage. (C'') Myopodia and growth cone filopodia intermingle to form the nascent synapse at 14:00 hours. (D) Quantification of myopodia contact stability. None of the myopodial contacts with the presumed non-partner neurons was stabilized. ***P=1.7x10-6; Fisher's test. Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. Dual-color imaging of pre- and postsynaptic structures. (A-D'') Images of myopodia (A-D,A'-D'; mGFP, green) and motor axons (A-D,A''-D''; anti-HRP:Cy5, magenta) in dissected embryos during 13:30-14:00 hours. Tips of myopodia (arrows) and ends of axons (arrowheads) are indicated. The asterisk indicates the lamellipodia-like structure (see text). Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. Distribution of CAPS during pre- and postsynaptic interaction. Triple-color imaging of CAPS-YFP in muscle (A-C,A'''-C'''; green), mCFP in muscle (A'-C',A'''-C'''; blue) and axon (A''-C'', A'''-C'''; anti-HRP:Cy5, magenta) in dissected embryos at 13:00-14:00 hours. (A-A''') CAPS-YFP accumulation at the tips of myopodia (arrowhead) before innervation. (B-B''') CAPS-YFP concentrates at the tips of myopodia (arrowhead) even after growth cones contact and extend along myopodia (arrows). (C-C''') No concentration of CAPS-YFP is seen in assembled myopodia (asterisk). Scale bar: 5 µm.

 

Figure 8
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  		Fig. 8. Synaptogenesis in caps and caps trn mutants. (A) Interaction between myopodia and motoneuronal growth cones in control, capsC28fs/Df(3L)Ly, trn25/4/capsC28fs trndelta17 and capsC28fs trndelta17at 13:30 hours. Scale bar: 10 µm. (B) Quantification of numbers of contact points between myopodia and growth cones at 13:30 hours AEL. Numbers of contacts are reduced in capsC28fs/Df(3L)Ly, trn25/4/capsC28fs trndelta17 and capsC28fs trndelta17. **P<0.01, ***P<0.001; two-tailed t-test. (C) Fraction of hemisegments that have formed a nascent terminal at 14:00 hours AEL. **P<0.01, *** P<0.001; {chi}2 test. We define a nascent terminal as a bifurcated structure formed on lamellipodia-like myopodial cluster (as shown in Fig. 6C) or on the body of M12 (as shown in Fig. 6D). The analysis was undertaken in a genotype-blinded manner. (D-G) Size of the M12 axon terminal at 15:00 hours is reduced in caps and caps trn mutants. (D) Axon terminals visualized with anti-HRP immunostaining in wild-type, capsC28fs/Df(3L)Ly, trn25/4 and capsC28fs trndelta17 embryos at 15:00 hours. The white arrow and line indicate the length of the axon terminal and muscle, respectively, that were used for the quantitative analyses in E. Scale bar: 10 µm. (E) Terminal size is reduced in caps and caps trn mutants. The ratio of the length of MN12 axon terminals to that of M12 in controls (+/+, n=185 segments; +/Df(3L)Ly, n=77), caps mutants (capsC28fs/Df(3L)Ly, n=98; capsL259fs/Df(3L)Ly, n=123), trn mutants (trn25/4, n=62) and caps trn mutants (capsC28fs, trndelta17, n=51; caps65.2, trnS064117, n=92). The ratio is significantly smaller in two alleles each of caps, trn and caps trn mutants as compared with +/+ controls (*P<0.05, **P<0.01, ***P<0.001; two-tailed t-test). The same statistical results are obtained when +/Df(3L)Ly line was used as a control instead of +/+ for the t-test (not shown). The ratios in caps trn double mutants (capsC28fs, trndelta17 or caps65.2, trnS064117) are significantly smaller than in caps (capsC28fs/Df(3L)Ly) or trn (trn25/4) single mutants. The same statistical results are obtained when the double mutants are compared with capsL259fs/Df(3L)Ly, another caps single mutant line (not shown). (F) Terminal formation is normal in caps-negative muscles, M6 and M7. No significant difference in the size of M6/7 terminals in capsC28fs/Df(3L)Ly (0.40; n=47) and capsC28fs, trndelta17 (0.36; n=47) compared with controls (0.35; n=47) (P>0.05; two-tailed t-test). (G) Postsynaptic expression of a deletion form of CAPS lacking the intracellular domain affects axon terminals on M12. Graph shows ratio of the length of MN12 axon terminals to that of M12 in control embryos and in embryos expressing a deletion mutant form of CAPS (CAPS-ID) in postsynaptic muscles (M12>capsID) or in presynaptic neurons (elav>capsID). Terminal size is reduced in M12>capsID (0.28; n=30) but not in elav>capsID (0.38; n=35) compared with controls (0.36; n=134; ***P<0.001; two-tailed t-test). Error bars in E-G represent the mean±s.e.m.

 

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© The Company of Biologists Ltd 2009